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Proteolytic depilation of lambskins

A thesis presented in partial

fulfilment of the requirements

for the degree of

Doctor of philosophy in Bioprocess Engineering at Massey University, Palmerston North, New Zealand

Richard Edmonds

2008

i

Abstract

The processing of lambskins plays an important role in the New Zealand meat industry.

The use of enzyme dewooling offers considerable advantages over the conventional

depilation method which generates unpleasant working conditions and poses product

quality risks when not properly handled. Prior to this work it was unclear from the

literature why the practice of enzymatic depilation had not generally been adopted by

industry. The aim of this work was to determine the problems associated with enzymatic

depilation and provide a mechanistic understanding of the dewooling and damaging

processes of enzyme depilation to provide underpinning knowledge for the design of a

successful enzymatic depilation system.

It was found that variability in depilation between different regions of the skin resulted

in either over exposure of the skin to the enzyme regent and subsequent damage or

underexposure of the skin to the enzyme reagent and incomplete depilation. Two

approaches were taken in the work: Firstly an attempt was made for the first time to

understand the variability in enzymatic depilation so that the variability observed in

enzymatic depilation could potentially be reduced, thereby allowing a complete

depilation process with no overexposure. Secondly an investigation was made for the

first time to understand the cause of damage to skins during the process of enzymatic

depilation so that the enzyme depilation process could potentially be modified to avoid

damage.

Experimental work characterising the time course of depilation and damage

development was carried out and compared with the variation of physical properties

across the skin. Correlations between depilation and physical properties such as

thickness, grease content and follicle density were found. Reduction in the variability of

these properties would likely improve the evenness of depilation but would not reduce it

enough to eliminate damage due to over exposure.

A range of techniques including: immunohistology, 2-dimensional electrophoresis,

matrix assisted laser desorption ionisation, and atomic force microscopy were used to

probe the structural and biochemical mechanism of enzyme depilation and damage. In

this way it was found that the removal of minor collagen components were the likely

cause of damage observed. In particular the removal of collagen VI was associated with

a disruption of the smooth mesh of fine collagen fibres observed at the surface of the

leather.

The key requirement identified for a successful enzyme depilation system was the use

of a broad spectrum protease which has no activity against collagen VI. The means to

select a protease with these attributes was also developed by adopting a micro depilation

assay incorporating immunohistology. This knowledge will enable the future

development of non damaging enzyme depilatory reagents that could revolutionise the

industry.

R.L. Edmonds

ii

Acknowledgments

Once in a while a genius comes along and astonishes the world with their

accomplishments. I am not one of those people, but I have had the opportunity to work

with some astonishing individuals in the preparation of this thesis. The work could not

have been accomplished without the help of the following:

Firstly I need to acknowledge the financial support from the foundation for research

science and technology (FRST) under the program LSRX0301 through the New

Zealand Leather and Shoe Research Association (LASRA) and Tony Passman in

particular without whom the project would not have been made available in the first

place.

My supervisors in alphabetical order; Timothy Allsop, John Bronlund, Yusuf Chisti,

and Gill Norris. Without their guidance this thesis and the work it is based on would

have been concise to the point of inconsequence.

Santanu DebChoudhury – thanks for your help with preparation and running of the

electrophoresis, the sections involving those very demanding techniques would not have

been possible without you. Thanks also to the other members of the Massey Lab who

gave their assistance.

Thanks to all the technical staff at LASRA and Katherine Coolbear-Stern in particular –

thanks for providing assistance in processing the leather in the tannery and in

measurements of physical tests. Thank you also to George particularly for obtaining

some of the more unusual fine chemicals that I used in the work.

Merve Birtles and all those people in the Institute of veterinary and biological sciences

histology preparation laboratory – thanks for letting me use your flash equipment and

your much needed expertise. The immuno-histology parts of the thesis were only made

possible through your assistance

Steven Werner – Thanks for suffering all the pitfalls and fishhooks of an Engineering

type PhD before me so that I could have some chance to avoid them myself.

Last but not least I need to thank my family, and personal support from my wife Tracy.

For the most part they want to say they know someone called Dr Dick but they mean

well all the same.


iii

Success is the ability to go from one failure to another with no loss of enthusiasm

Sir Winston Churchill (1874 – 1965)

If you’re not part of the solution you’re part of the precipitate

Anon

R.L. Edmonds

iv

Table of contents

Abstract .......................................................................................................................... i

Acknowledgments......................................................................................................... ii

Table of contents.......................................................................................................... iv

Index of figures ......................................................................................................... xvii

Index of tables......................................................................................................... xxvii

Preface: Thesis overview ......................................................................................... xxix

Glossary .................................................................................................................... xxx

INTRODUCTION.............................................................................1

1.1 Definition of the problem ................................................................................... 1

1.2 Aims and objectives ............................................................................................ 2

1.3 Approach.............................................................................................................. 2

LITERATURE REVIEW.................................................................3

2.1 Introduction......................................................................................................... 3

2.2 Conventional process of fellmongery ................................................................ 3

2.2.1 Flaying .......................................................................................................... 3

2.2.2 Pre-fleshing ................................................................................................... 4

2.2.3 Painting ......................................................................................................... 4

2.2.3.1 Mechanism of sulfide depilation............................................................... 5

2.2.4 Potting (stacking) .......................................................................................... 7

2.2.5 Pulling ........................................................................................................... 7

2.2.6 Liming........................................................................................................... 7

2.2.7 Delime........................................................................................................... 8

2.2.8 Bating ............................................................................................................ 8

2.2.9 Pickle............................................................................................................. 9

2.2.10 Problems associated with conventional depilation ....................................... 9

2.2.11 Conventional processing - Summary ............................................................ 9


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2.3 Enzymatic depilation as an alternative to the conventional process ............10

2.4 Other alternatives to sulfide depilation and enzyme depilation ...................11

2.4.1 Plucking wool ............................................................................................. 12

2.4.2 Shearing....................................................................................................... 12

2.4.3 Water jet and laser shearing ........................................................................ 12

2.4.4 Cryogenic depilation ................................................................................... 13

2.4.5 Scalding....................................................................................................... 13

2.4.6 Laser hair disruption ................................................................................... 13

2.4.7 Electro-depilation........................................................................................ 14

2.4.8 Chaotropic agents........................................................................................ 14

2.4.9 Chemical shearing....................................................................................... 14

2.4.10 Alternative chemical depilation agents ....................................................... 15

2.4.10.1 Wholly alkali depilation......................................................................15

2.4.10.2 Oxidative depilation............................................................................16

2.4.10.3 Acid depilation ....................................................................................16

2.4.10.4 Other organo-compounds....................................................................17

2.4.11 Alternative depilation methods - Summary ................................................ 17

2.5 Skin microstructure ..........................................................................................18

2.5.1 Extracellular matrix (ECM) ........................................................................ 20

2.5.2 Basal membrane .......................................................................................... 21

2.5.3 Epidermis .................................................................................................... 22

2.5.3.1 Epidermal structure .................................................................................22

2.5.3.2 Epidermal cell adhesion ..........................................................................23

2.5.4 Hair structure............................................................................................... 26

2.5.4.1 Hair shaft structure..................................................................................26

2.5.4.1.1 The medulla.......................................................................................27

2.5.4.1.2 The cortex..........................................................................................27

2.5.4.1.3 The cuticle.........................................................................................27

2.5.4.2 Hair root structure ...................................................................................28

2.5.4.2.1 Inner root sheath (IRS)......................................................................28

2.5.4.2.2 Outer root sheath (ORS)....................................................................28

2.5.4.2.3 Companion layer ...............................................................................28

2.5.4.3 Hair growth .............................................................................................28

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2.5.4.3.1 Keratinisation .................................................................................... 29

2.5.4.4 Connective tissue sheath (follicular basement membrane)..................... 30

2.5.5 Skin microstructure - Summary .................................................................. 30

2.6 Skin Components .............................................................................................. 32

2.6.1 Collagen ...................................................................................................... 32

2.6.1.1 The collagen triple helix ......................................................................... 33

2.6.1.2 Fibre forming collagens .......................................................................... 33

2.6.1.3 Fibril associated collagen with interrupted triple helixes (FACIT) ........ 35

2.6.1.4 Short chain collagens .............................................................................. 37

2.6.1.5 Basement membrane collagens............................................................... 37

2.6.1.6 Multiplexins ............................................................................................ 38

2.6.1.7 Collagens with trans-membrane domains (MACIT) .............................. 38

2.6.1.8 Other collagens ....................................................................................... 39

2.6.2 Glycoproteins .............................................................................................. 41

2.6.2.1 Fibronectin .............................................................................................. 41

2.6.2.2 Fibulin ..................................................................................................... 43

2.6.2.3 Laminin ................................................................................................... 43

2.6.2.4 Entactin/Nidogen .................................................................................... 44

2.6.2.5 Glycoproteins in hair............................................................................... 44

2.6.3 Polysaccharides ........................................................................................... 44

2.6.3.1 Glycosaminoglycans ............................................................................... 44

2.6.3.1.1 Hyaluronic acid ................................................................................. 46

2.6.3.1.2 Dermatan sulfate ............................................................................... 46

2.6.3.2 Glycogen ................................................................................................. 47

2.6.4 Proteoglycans .............................................................................................. 47

2.6.4.1 The role of proteoglycans in skin............................................................ 48

2.6.4.2 Specific proteoglycans ............................................................................ 49

2.6.4.2.1 Biglycan ............................................................................................ 49

2.6.4.2.2 Decorin.............................................................................................. 50

2.6.4.2.3 Versican ............................................................................................ 50

2.6.5 Other Proteins ............................................................................................. 50

2.6.5.1 Elastin...................................................................................................... 50

2.6.5.2 Keratin..................................................................................................... 52


vii

2.6.5.3 Tenascin ..................................................................................................52

2.6.5.4 Vitronectin...............................................................................................53

2.6.5.5 Trichohyalin ............................................................................................53

2.6.6 Lipids .......................................................................................................... 53

2.6.7 Skin components - Summary ...................................................................... 53

2.7 The enzyme depilation mechanism..................................................................54

2.7.1 Structural aspects of enzyme depilation...................................................... 55

2.7.1.1 Diffusion through skin ............................................................................55

2.7.1.2 Stages of depilation .................................................................................56

2.7.1.3 Impact of enzyme depilation on macromolecular structures ..................56

2.7.1.3.1 Basal layer.........................................................................................56

2.7.1.3.2 Cell dispersion...................................................................................57

2.7.1.3.3 Root sheaths ......................................................................................57

2.7.2 Chemical mechanism of enzyme depilation ............................................... 57

2.7.2.1 Specific proteins and the enzyme depilation mechanism .......................58

2.7.2.1.1 Collagen types I and III.....................................................................58

2.7.2.1.2 Collagen type IV ...............................................................................59

2.7.2.1.3 Keratin...............................................................................................59

2.7.2.1.4 General proteases ..............................................................................60

2.7.2.2 Carbohydrates .........................................................................................61

2.7.2.2.1 Glycosaminoglycans – Hyaluronic acid............................................62

2.7.2.2.2 Glycosaminoglycans – Dermatan sulfate..........................................62

2.7.2.2.3 Glycoproteins ....................................................................................63

2.7.2.3 Influence of pH (alkali in particular) ......................................................63

2.7.2.4 Salts .........................................................................................................64

2.7.3 Enzyme assisted depilation ......................................................................... 64

2.7.4 Ideal requirements....................................................................................... 65

2.7.5 Enzyme depilation mechanism – Summary................................................ 65

2.8 Protease specificity and inhibition...................................................................66

2.8.1 Proteases and inhibition: By protease class ................................................ 67

2.8.1.1 Serine Proteases ......................................................................................68

2.8.1.2 Cysteine Proteases...................................................................................69

2.8.1.3 Aspartic Acid Proteases ..........................................................................69

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2.8.1.4 Metallo-proteases .................................................................................... 70

2.8.1.4.1 Metzincins......................................................................................... 70

2.8.1.5 Non-specific protease inhibitors ............................................................. 71

2.8.1.5.1 Multi-class protease inhibitors .......................................................... 71

2.8.1.5.2 Metal chelating substances................................................................ 71

2.8.1.5.3 Indirect reduction in unwanted protease activity .............................. 72

2.8.2 Proteases and inhibition: By protease substrate .......................................... 72

2.8.2.1 General extracellular matrix specific exogenous proteases .................... 72

2.8.2.2 Collagenase ............................................................................................. 74

2.8.2.2.1 Collagenase inhibitors....................................................................... 76

2.8.2.3 Keratinase................................................................................................ 76

2.8.2.4 Elastase.................................................................................................... 77

2.8.2.5 Glycosidase ............................................................................................. 77

2.8.2.6 Minor components of the extracellular matrix proteolysis ..................... 77

2.8.3 Protease specificity and inhibition – Summary........................................... 78

2.9 Literature review – Conclusions...................................................................... 79

EXPERIMENTAL CHARACTERISATION OF DEPILATION

...........................................................................................................81

3.1 Introduction....................................................................................................... 81

3.2 Enzyme depilation............................................................................................. 81

3.2.1 Initial experiments....................................................................................... 82

3.2.1.1 Table of enzymes sourced for this work ................................................. 82

3.2.1.2 Enzyme application method.................................................................... 82

3.2.1.3 Depilation results .................................................................................... 84

3.2.1.4 Wool product........................................................................................... 84

3.2.1.4.1 Methodology ..................................................................................... 84

3.2.1.4.2 Alkali solubility................................................................................. 85

3.2.1.4.3 Acid solubility................................................................................... 85

3.2.1.5 Results from the solubility trials ............................................................. 86

3.2.1.6 SEM (Scanning electron microscopy) of enzyme treated wool.............. 87

3.2.1.7 Grain surface ........................................................................................... 89


ix

3.2.2 Selected enzyme purification ...................................................................... 91

3.2.2.1 Low pH native PAGE separation method...............................................92

3.2.2.2 Low pH native PAGE separation of enzymes.........................................94

3.2.3 Enzyme selection – Summary..................................................................... 98

3.3 Characterisation of optimal enzyme depilatory .............................................99

3.3.1 Assay method.............................................................................................. 99

3.3.1.1 Skin powder azure substrate (SPA) ........................................................99

3.3.1.2 Skin powder azure assay .......................................................................100

3.3.1.3 Skin powder azure assay results............................................................100

3.3.1.4 Assay error ............................................................................................104

3.3.2 Effect of pH............................................................................................... 104

3.3.2.1 pH effect on measured activity .............................................................104

3.3.2.2 pH effect on depilatory effectiveness....................................................106

3.3.2.3 Quenching enzyme activity...................................................................106

3.3.3 Effect of temperature................................................................................. 110

3.3.4 System optimisation - Summary ............................................................... 112

3.4 Enzyme characterisation - Conclusions ........................................................113

VARIATION IN ENZYMATIC DEPILATION....................... 114

4.1 Introduction.....................................................................................................114

4.2 Heterogeneity of depilation ............................................................................115

4.2.1 Depilation method..................................................................................... 115

4.2.1.1 Depilation progression results...............................................................115

4.2.2 Grain surface damage................................................................................ 120

4.3 Variability of the skin (Skin heterogeneity)..................................................127

4.3.1 Approach ................................................................................................... 127

4.3.2 Variable properties .................................................................................... 129

4.3.2.1 Skin structure variation .........................................................................130

4.3.2.2 Variation in thickness............................................................................134

4.3.2.3 Grease content.......................................................................................137

4.3.2.4 Fibre diameter .......................................................................................140

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4.3.2.5 Follicle distribution ............................................................................... 142

4.3.2.6 Wool root strength ................................................................................ 143

4.3.3 Skin variation – Summary......................................................................... 145

4.4 Impact of washing on soluble proteins from the skin.................................. 146

4.4.1 Initial washing proof-of-concept trial ....................................................... 146

4.4.1.1 Method .................................................................................................. 147

4.4.1.2 Results................................................................................................... 148

4.4.1.2.1 Changes in depilation from washing............................................... 148

4.4.1.2.2 Skin structure .................................................................................. 150

4.4.1.2.3 Protein removal ............................................................................... 150

4.4.1.2.4 Crust leather .................................................................................... 151

4.4.2 Detailed washing experiment.................................................................... 156

4.4.2.1 Method .................................................................................................. 156

4.4.3 Removal of soluble proteins - Summary................................................... 157

4.5 Variability of enzyme depilation – Conclusions........................................... 158

COMPONENT REQUIREMENTS FOR ENZYMATIC

DEPILATION ...............................................................................159

5.1 Introduction..................................................................................................... 159

5.2 The structural mechanism of enzyme depilation ......................................... 159

5.2.1 Initial investigation of the enzyme depilation mechanism........................ 159

5.2.2 Enzyme depilation mechanism – SACPIC results .................................... 160

5.2.3 Structural modification – Summary.......................................................... 162

5.3 Glycosaminoglycan changes during enzyme depilation .............................. 163

5.3.1 Method ...................................................................................................... 163

5.3.2 Results....................................................................................................... 164

5.3.3 Glycosaminoglycans changes - Summary ................................................ 167

5.4 Individual protein changes during depilation .............................................. 167

5.4.1 Immunohistology methods and materials ................................................. 169

5.4.2 SACPIC controls....................................................................................... 172


xi

5.4.3 Location of skin minor proteins and their presence in conventionally

processed material results ......................................................................................... 176

5.4.3.1 Location of laminin and its response to conventional processing.........176

5.4.3.2 Location of collagen VI and its response to conventional processing ..178

5.4.3.3 Location of collagen VII and its response to conventional processing.180

5.4.3.4 Location of fibronectin and response to conventional processing ........183

5.4.3.5 Location of elastin and response to conventional processing ...............185

5.4.3.6 Conclusions for the location of minor proteins.....................................188

5.4.4 Minor proteins in enzyme depilated skins ................................................ 189

5.4.4.1 Changes to fibronectin during enzymatic depilation ............................189

5.4.4.2 Changes to elastin during enzymatic depilation....................................191

5.4.4.3 Changes to collagen VI during enzymatic depilation ...........................194

5.4.4.4 Changes to collagen VII during enzymatic depilation..........................197

5.4.4.5 Response of laminin during enzymatic depilation ................................199

5.4.5 Examination of specificity of immunohistology by Western blotting...... 201

5.4.5.1 Sample preparation for electrophoresis method....................................202

5.4.5.2 Western blotting method.......................................................................202

5.4.5.3 Western blotting results.........................................................................204

5.4.6 Individual protein changes during depilation - Summary......................... 207

5.5 Determining the reason for damage to surface proteins .............................208

5.5.1 Surface protein investigation method ....................................................... 209

5.5.1.1 Surface protein investigation reagents ..................................................209

5.5.1.2 Initial sample generation .......................................................................210

5.5.1.3 Atomic force microscopy method.........................................................212

5.5.1.4 Electrophoresis method.........................................................................212

5.5.1.5 Preparation for matrix assisted laser desorption ionisation time of flight

analysis 213

5.5.1.6 MALDI TOF analysis method ..............................................................214

5.5.2 Results ....................................................................................................... 214

5.5.2.1 Atomic force microscopy......................................................................214

5.5.2.2 2D SDS PAGE......................................................................................220

5.5.2.3 MALDI TOF .........................................................................................222

5.5.3 Stratified samples – Summary .................................................................. 228

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xii

5.6 Determination of the nature of changes in the proteins – Conclusions...... 229

CONTROLLING UNDESIRABLE ENZYME ACTIVITY ....231

6.1 Salt addition..................................................................................................... 231

6.1.1 Determination of activity against general skin substance......................... 232

6.1.1.1 Method .................................................................................................. 232

6.1.2 Salt depilation method .............................................................................. 234

6.1.3 Depilation results ...................................................................................... 235

6.1.4 The effect of sodium chloride on grain structure...................................... 238

6.1.5 Alternative salts......................................................................................... 240

6.1.6 Method ...................................................................................................... 240

6.1.7 Salt solution formulation........................................................................... 241

6.1.8 Batch trial methodology............................................................................ 242

6.1.9 Results....................................................................................................... 242

6.1.10 Addition of salt – Summary...................................................................... 245

6.2 Enzyme Specificity .......................................................................................... 245

6.2.1 Single section depilation method SACPIC visualisation.......................... 246

6.2.2 Single section depilation method development ........................................ 247

6.2.2.1 Chemical fixation.................................................................................. 248

6.2.3 Immunohistology of enzyme treated single sections ................................ 248

6.2.3.1 Single section depilation immunohistology method development ....... 249

6.2.3.2 Single section enzyme application method........................................... 249

6.2.3.3 Single section SACPIC method ............................................................ 250

6.2.3.4 Single section immunohistology method.............................................. 250

6.2.4 Single section depilation ........................................................................... 250

6.2.5 Results....................................................................................................... 251

6.3 Controlling undesirable activity - Conclusions ............................................ 254

CONCLUSIONS: PROTEOLYTIC DEPILATION OF

LAMBSKINS.................................................................................256

7.1 Research overview (The big picture)............................................................. 256


xiii

7.2 Future direction of further research .............................................................257

APPENDICES............................................................................... 260

8.1 Published works ..............................................................................................260

8.2 Standard conventional processes ...................................................................261

8.2.1 Standard conventional painting and liming process.................................. 261

8.2.2 Standard delime and bate process ............................................................. 261

8.2.3 Standard pickle.......................................................................................... 262

8.2.4 Aqueous degrease...................................................................................... 262

8.2.5 Chrome tannage ........................................................................................ 262

8.2.6 Retan to crust leather................................................................................. 263

8.3 Stain solutions..................................................................................................263

8.3.1 Section preparation.................................................................................... 263

8.3.1.1 Cryostat frozen samples ........................................................................263

8.3.1.2 Formalin fixation...................................................................................264

8.3.1.2.1 Standard buffered formalin solution ...............................................264

8.3.1.2.2 Buffered formalin in brine solution ................................................264

8.3.1.3 Bouin’s fluid fixation ............................................................................264

8.3.1.4 Paraffin embedding protocol.................................................................264

8.3.1.5 De wax protocol ....................................................................................265

8.3.2 Phosphate buffered saline 0.01M (PBS) ................................................... 265

8.3.3 SACPIC staining method .......................................................................... 265

8.3.3.1 Celestin blue..........................................................................................265

8.3.3.2 Scott’s tap water....................................................................................266

8.3.3.3 Safranine solution..................................................................................266

8.3.3.4 Picric acid in ethanol.............................................................................266

8.3.3.5 Picro-indigo-carmine.............................................................................266

8.3.3.6 SACPIC staining protocol.....................................................................266

8.3.4 Alcian blue periodic acid Schiff stain protocol......................................... 267

8.3.5 Mounting protocol..................................................................................... 267

8.3.6 Immuno-histological stain protocol .......................................................... 267

8.3.7 Epitope/Antigen retrieval methods ........................................................... 268

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8.3.7.1 Citrate buffer epitope retrieval .............................................................. 268

8.3.7.1.1 Citrate buffer solution ..................................................................... 268

8.3.7.1.2 Citrate buffer epitope retrieval method........................................... 269

8.3.7.2 Trypsin epitope retrieval solutions........................................................ 269

8.3.7.2.1 Trypsin stock solution (0.5% in distilled water) ............................. 269

8.3.7.2.2 Calcium chloride stock solution (1% in distilled water)................. 269

8.3.7.2.3 Trypsin working solution (0.05% in distilled water) ...................... 269

8.3.7.2.4 Trypsin epitope retrieval method .................................................... 269

8.4 Polyacrylamide gel methods........................................................................... 270

8.4.1 Isocratic gel (7.5% Polyacrylamide, 2 small format gels, example)......... 270

8.4.2 Gradient gel (7 – 14 % Polyacrylamide, 2 small format gels, example) .. 270

8.4.2.1 Light fraction......................................................................................... 271

8.4.2.2 Heavy fraction....................................................................................... 271

8.4.3 Ornstein Davis native gel .......................................................................... 271

8.4.4 Low pH native PAGE gel ......................................................................... 272

8.5 Polyacrylamide electrophoresis ..................................................................... 272

8.5.1 Sample extraction buffer ........................................................................... 272

8.5.2 Chloroform-Methanol precipitation method............................................. 273

8.5.3 1D Polyacrylamide sample buffer............................................................. 274

8.5.4 10x SDS tank buffer.................................................................................. 274

8.6 2D Polyacrylamide electrophoresis ............................................................... 274

8.6.1 Sample preparation ................................................................................... 274

8.7 Poly acrylamide gel staining........................................................................... 275

8.7.1 Fixatives .................................................................................................... 275

8.7.1.1 SDS PAGE fixative............................................................................... 275

8.7.1.2 Native PAGE fixative ........................................................................... 275

8.7.2 Silver stain................................................................................................. 275

8.7.2.1 Sensitising agent ................................................................................... 275

8.7.2.2 Silver stain............................................................................................. 276

8.7.2.3 Developer .............................................................................................. 276

8.7.2.4 Stop solution ......................................................................................... 276

8.7.2.5 Silver staining protocol ......................................................................... 276


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8.7.3 Coomassie blue stain................................................................................. 276

8.7.4 Western Blotting ....................................................................................... 277

8.7.4.1 10x Transfer Buffer...............................................................................277

8.7.4.2 1x Transfer Buffer.................................................................................277

8.7.4.3 10x Phosphate Buffered saline for Western (10x PBSW) .....................277

8.7.4.4 1x Phosphate Buffered saline for Western (1x PBSW) .........................278

8.7.4.5 BSA diluent for Western (BSAW)........................................................278

8.7.4.6 Western transfer protocol......................................................................278

8.7.4.7 Western immuno probe protocol...........................................................278

8.7.4.8 Western staining protocol .....................................................................279

8.8 Document statistics..........................................................................................279

REFERENCES.............................................................................. 280

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xvii

Index of figures

Figure 1: Reduction of keratin by sulfide reaction .......................................................6

Figure 2: Diagram of skin showing structures important for leather making

(Sharphouse, 1971). ................................................................................................18

Figure 3: Simple raw skin micro structure medical definitions..................................19

Figure 4: Leather maker’s definitions of the different parts of the dermis.................19

Figure 5: Simple skin macrostructure (from (Dempsey, 1984)).................................20

Figure 6: Hair structure (coloured for sake of visualisation)......................................26

Figure 7: Schematic magnification of collagen fibre .................................................34

Figure 8: Schematic of collagen weave cross-section (in general the fibres lie parallel

to skin surface) ........................................................................................................34

Figure 9: Fibril associated collagen. Note that a fibril associated collagen interacts

with a whole fibril of collagen molecules for a portion of its single molecular

length leaving a portion of the FACIT molecule exposed to the extra molecular

medium....................................................................................................................36

Figure 10: Type IV collagen quaternary structure from ((Timpl et al., 1981); (von

Loutis et al., 1984),(Stanley et al., 1982)) ..............................................................38

Figure 11: Hierarchy of collagen VI structure..........................................................39

Figure 12: Type VII collagen quaternary structure ..................................................40

Figure 13: Schematic of laminin based on rotary shadowing images (Sasaki & Timpl,

1999). ......................................................................................................................43

Figure 14: Hyaluronic acid disaccharide unit...........................................................46

Figure 15: Dermatan sulfate disaccharide unit .........................................................46

Figure 16: Cross linking amino acids of elastin .......................................................51

Figure 17: Effect of the enzyme depilation processes on wool, a higher score

indicates wool that is more susceptible to the solubility test and hence may be

damaged. 86

Figure 18: SEM image of greasy wool control, note the cuticle of the wool fibre is

visible. 88

Figure 19: SEM of enzyme treated wool sample, note the defined cuticle indicating

that the wool fibre is not damaged by immersion in a buffered enzyme depilation

solution after a wash in a neutralising scour. ..........................................................88

Figure 20: Optical microscopy of grain surface of pelts processed through to crust

leather using the conventional lime/sulfide based process. Note the regular

R.L. Edmonds

xviii

appearance of the follicle mouths and smoothness of the interfollicular pads. (The

bar is 500 um) (a black arrow indicates an interfollicular pad (hill), a white arrow

indicates the valley between two interfollicular pads)............................................ 89

Figure 21: Optical microscopy images of the effect of enzyme process on grain surface

(bar is 500 um, all images are at the same magnification)...................................... 90

Figure 22: Poly acrylamide electrophoresis separation of enzymes under low pH (4.3)

conditions using 10% polyacrylamide concentration. Lanes are numbered along the

bottom and equivalent excised gel strip indicated on the right. Lanes were as

follows: Batinase ® Neutral, Purafect 4000L, Protease 6L, Protease 200, Promod,

Bromelain................................................................................................................ 94

Figure 23: General skin substance assay. A comparison of duplicate tests using

Purafect for each of the experimental substrates was examined........................... 101

Figure 24: Skin powder azure assay regression The original raw absorbance data is

shown as black triangles. The predicted plot from the equation with two terms is

show as a dashed black line The prediction using the three term regression

equation is shown in grey...................................................................................... 102

Figure 25: Skin powder azure assay for initial slope estimation of the SPA assay using a

non linier fit. The original raw absorbance data is shown as black triangles. The

prediction using the non-linear regression is shown in grey. The predicted initial

slope is then illustrated by the black line. ............................................................. 103

Figure 26: Effect of pH on the skin powder azure assay (Purafect)....................... 105

Figure 27: Depilation pH response......................................................................... 106

Figure 28: Influence of low pH on enzyme activity against general skin substance as

measured by the skin powder azure test................................................................ 107

Figure 29: Regeneration of activity as measured at pH 10.5 after 5 seconds exposure

to low pH, x axis represents the exposure pH, y axis represents the activity of the

enzyme at pH 10.5 ................................................................................................ 108

Figure 30: Regeneration of activity in pH 10.5 after 2 hours exposure to low buffer

pH, x axis represents the exposure pH, y axis represents the activity of the enzyme

at pH 10.5.............................................................................................................. 109

Figure 31: Regeneration of activity after 24 hours exposure to low pH, x axis

represents the exposure pH, y axis represents the activity of the enzyme at pH 10.5

110

Figure 32: Effect of temperature on enzyme activity ............................................. 111


xix

Figure 33: Transformed rate vs. temperature plot indicating the activation energy of

the enzyme. Assuming an Arrhenius relationship the slope is –E/R where: R is the

gas constant (8.314 J.mol-1.K-1), E is the activation energy (63000 J.mol-1)

(Levenspiel, 1999).................................................................................................111

Figure 34: Progression of enzymatic depilation. The first regions to depilate are the

flanks. As depilation proceeds the regions of depilated area grow in size until the

back bone is all that is left. The backbone and neck depilate last.........................116

Figure 35: Enzyme depilated skin: beginning at 0hrs with 1hr intervals. The results

from the duplicate skin match the results observed for the illustrated skin (the holes

in the skin are sample holes) Note depilation in flanks first (arrowed) ................119

Figure 36: Region of undamaged skin surface (red arrows) located between non-

depilated skin (green arrow) and damaged skin (blue arrows) (bar is 200 um).

Damage becomes progressively worse the further away from the non-depilated

region (not shown) ................................................................................................125

Figure 37: Typical damage observed on the surface of enzyme depilated skins at a

distance at least 5 cm away from the non-depilated regions (bar is 200 um). ......126

Figure 38: Conventionally processed skin from the neck region (bar is 200 um)..126

Figure 39: Sample sites. The relative location of each of the 41 sample positions are

marked with an X. .................................................................................................128

Figure 40: Enzyme depilation time. Enzyme depilation was shown to take longer

down the mid line of the pelt, in particular in the neck area (top). The first

depilation occurred in the middle of the flanks.....................................................128

Figure 41: Fibre direction, adapted from Haines (2006). .......................................131

Figure 42: Left: Stiffness as measured by bendability, adapted from Lange et al,

(2002). Sample positions are marked with an x. Note stiffness in the backbone.

Right: Correlation of bending stiffness with enzyme depilation time. Note the good

correlation p<0.001. The confirmation confirmation of a possible curvilinear

relationship is left for future researchers...............................................................133

Figure 43: Left: Stiffness as measured by percent extension, adapted from Lange et

al, (2002). Note the greater stiffness found towards the midline and less stiffness

found in the flanks. Right: Correlation of bending stiffness with enzyme depilation

time. Note the correlation (p=0.004).....................................................................133

Figure 44: Left: Determination of the crust leather thickness. Note the thicker region

down the back bone and in the butt area. This also corresponds to the same general

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xx

pattern of thickness noted by previous authors (Cooper, 1998b). Right: Correlation

of thickness with enzyme depilation time, note good correlation (p<0.001)........ 135

Figure 45: Left: Distribution of the grain thickness. Note the thicker grain in the butt

region. Right: Correlation of grain thickness to enzyme depilation time, Note the

good correlation (p<0.001) ................................................................................... 136

Figure 46: Left: Distribution of the corium thickness. Note the thicker grain in the

butt region. Right: Correlation of corium thickness to enzyme depilation time, Note

the good correlation (p<0.001).............................................................................. 136

Figure 47: Left: Grease content of the full thickness of skin. Note the greater

quantity of grease located in the midline and butt region. Right: Correlation of total

grease to enzyme depilation time, note their was little correlation between the total

grease and the depilation time indicating that the fat within the structure of the skin

does not effect the depilation (p=0.04). ................................................................ 138

Figure 48: Left: Grease content in the skin surface as measured by surface darkness.

Note the darker regions at the mid line of the neck and butt. Right: Correlation of

surface grease content with depilation time, note the good correlation (p<0.001)

nb: darker regions are denoted by lower values for Lab ........................................ 140

Figure 49: Left: Distribution of fibre diameter. Note the greater fibre diameter in the

lower middle of the back. Right correlation of fibre diameter to enzyme depilation

time, note the lack of correlation (p>0.1).............................................................. 141

Figure 50: Left: Distribution of follicle density. Note the greater follicle density in

the centre of the back. Correlation of follicle density to enzymatic depilation time,

note the good correlation (p<0.001)...................................................................... 142

Figure 51: Left: Force per wool fibre. Note a slight increase in force per fibre in the

lower middle of the back. Right : Correlation of force per wool fibre with

enzymatic depilation time, note the lack of correlation (p>0.1). .......................... 143

Figure 52: Relationship between the forces required to remove a wool fibre from a

raw skin to the fibre diameter as measured at each of the 41 sites. There is a weak

correlation between the force required to pull out a wool fibre and the diameter of

that fibre (r2 =0.20 p=0.003) ................................................................................. 144

Figure 53: Comparison of the extent of enzymatic depilation after 16 hours from

washed vs. unwashed skins. Note the increase in the level of depilation when skins

are washed............................................................................................................. 149


xxi

Figure 54: Soluble protein removed during washing. Note that both the absolute

amount of soluble protein and the proportion of protein that was soluble decreased

after washing. ........................................................................................................150

Figure 55: Impact of washing on looseness............................................................152

Figure 56: The impact of washing on the softness of crust leather ........................153

Figure 57: Effect of washing on the quality of the grain surface ...........................154

Figure 58: Impact of washing on the strength of crust leather ...............................155

Figure 59: The effect of washing on the progress of enzyme depilation. Different

colours represent different washing times, The x-axis represents the time after

depilatory enzyme exposure. Note the improvement in the rate of depilation for

skins that were exposed to washing for longer periods.........................................156

Figure 60: Typical SACPIC stains of cross sections of skin (each image has the

same scale, bar is 50 um) ......................................................................................160

Figure 61: Alcian blue- Periodic acid Schiff staining of skin cross sections (both

images are at the same scale, bar is 100 um) Where: regions stained blue contain

carboxyl or sulfate groups, regions stained magenta contained material with glycol

units such as sugars and regions stained purple contain both. ..............................164

Figure 62: Immunohistological conceptual diagram modified from (Boenisch,

2001), a) antigen, b) primary antibody attached, c) biotinylated secondary antibody

attached, d) preformed streptavidin biotinylated horseradish peroxidase complex

attached. 171

Figure 63: SACPIC stain of thin cross sections (bar is 10 um). Collagen stained green,

cell nuclei stained black, and the inner root sheath is stained red. a) raw skin

clearly showing the inner root sheath in red and epidermal cells in black, b) Skin

exposed to Purafect for 8 hours showing removal of epidermal cells and most root

sheath cells. ...........................................................................................................173

Figure 64: Immunohistology of laminin in raw skin. Epidermal cells are visible at

the top (white arrow) and a wool root sheath visible on the bottom right, note the

positive staining occurring around the cells of the inner root sheath (black arrow)

(bar is 50 um). .......................................................................................................176

Figure 65: Immunohistology of laminin in a conventionally depilated slat with a

wool root visible as the vertical structure in the image centre, note again the

presence of positive staining around cells of the inner root sheath (arrowed)(bar is

50 um). 177

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xxii

Figure 66: Immunohistology of laminin in a conventionally processed skin after

deliming and bating. Note the positive staining in the cells of the root sheath

(arrowed) (bar is 50 um) ....................................................................................... 177

Figure 67: Immunohistology of laminin in a conventionally processed skin after

pickling. Note the positive staining in cells of the root sheath (arrowed) (bar is 50

um) 178

Figure 68: Immunohistology of collagen VI in raw skin with epidermis visible as

the pink cells at the top of the image and a wool fibre visible in the centre right of

the image (white arrow). Note the positive staining in the enamel region below the

epidermal cells (black arrow) (bar is 25 um) ........................................................ 179

Figure 69: Immunohistology of collagen VI in a conventionally depilated slat prior

to deliming and bating. Pieces of the epidermal layer have become free and can be

seen at the top of the image along with cornified cells (white arrow). Note the

positive staining in the enamel region (black arrow) (bar is 25 um) .................... 179

Figure 70: Immunohistology of collagen VI in a conventionally processed skin after

deliming and bating. Note the positive staining in the enamel region and down the

side of a wool root canal (black arrows). While the intensity of staining is less clear

there is clearly positive staining within the enamel region. (bar is 25 um) .......... 180

Figure 71: Immunohistology of collagen VI in a conventionally processed skin after

pickling. Note the positive staining in the enamel region (arrowed). While the

intensity of positive staining has decreased compared to the delimed and bated skin

there is positive staining within the enamel region. (bar is 25 um) ...................... 180

Figure 72: Immunohistology of collagen VII in raw skin. Note some positive

staining between some epidermal cells and the small plaques in the grain (arrowed)

not to be mistaken for the cells that are counter stained (bar is 25 um)................ 181

Figure 73: Immunohistology of collagen VII in conventionally depilated material.

Again positively stained plaques differentiable in the original sections were located

in the grain (arrowed). Unfortunately these are not differentiable from the

counterstained cells in the photomicrograph (bar is 20 um)................................. 181

Figure 74: Immunohistology of collagen VII in a conventionally processed skin

after deliming and bating. The positively stained plaques visible earlier in

processing cannot be seen in the delimed and bated material (bar is 20 um) ....... 182


xxiii

Figure 75: Immunohistology of collagen VII in a conventionally processed skin

after pickling. Note the positively stained plaques in the grain (arrowed) (bar is 20

um). 182

Figure 76: Immunohistology of fibronectin in raw skin. A wool root shaft is clearly

visible in the centre of the image with epidermal cells at the top and wool root

sheath cells descending into the wool root canal. Note the positive staining located

amongst the cells of the epidermis (arrowed). (bar is 20 um)...............................183

Figure 77: Immunohistology of fibronectin in conventionally depilated skin. Note

the lack of positive staining now that the epidermis is gone. Note that the

microscope light intensity was adjusted in an attempt to maximise any possible

staining. (bar is 20 um)..........................................................................................184

Figure 78: Immunohistology of fibronectin in conventionally delimed and bated

skin. Note the lack of positive staining now that the epidermis is gone. Note that

the microscope light intensity was adjusted in an attempt to maximise any possible


Figure 79: Immunohistology of fibronectin in conventionally processed pickled

pelt. Note the lack of positive staining now that the epidermis is gone. Note that the

microscope light intensity was adjusted in an attempt to maximise any possible


Figure 80: Immunohistology of elastin in raw skin, containing a wool root (white

arrow). Note the horizontal fibres in the grain and vertical fibres in the enamel

(black arrows) (bar is 100 um)..............................................................................186

Figure 81: Immunohistology of elastin in depilated skin. Note the fibres around a

collapsed blood vessel (black arrow), and the broken elastin fibres deeper in the

grain (white arrow). (bar is 100 um) .....................................................................186

Figure 82: Immunohistology of elastin in delimed skin. Note the horizontal fibres in

the grain and vertical fibres in the enamel (black arrows). Note also the presence of

broken fibres (white arrows). (bar is 100 um).......................................................187

Figure 83: Immunohistology of elastin in pickled pelt. Note the lack of staining in

some regions of the surface and the broken material scattered throughout. (bar is

100 um) 187

Figure 84: Western blot apparatus set-up of electro blotting sandwich .................203

Figure 85: Western blot ECL detection of collagen VI. Using anti-collagen VI

raised in rabbit (Chemicon AB7822). Pre-stained markers were used to define the

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molecular weight distribution (Blue ® plus L pre-stained standard cat LC5925,

invitrogen). Different bands represent different alpha chains including the highly

glycosilated alpha3 chain. ..................................................................................... 205

Figure 86: 7% SDS PAGE gel of skin extracts stained with coomassie blue.

Unstained markers were used to define the molecular weight distribution

(Fermentas #SM0430). Lanes 1,2 and 3 contain 1,2, and 4 uL of raw skin extract

respectively,. Lanes 4 and 5 contain 2.5 uL and 5 uL of mixed collagen

respectively. .......................................................................................................... 205

Figure 87: SDS PAGE and Western blot of skin proteins 7.5% Polyacrylamide gel.

Pre-stained markers were used to define the molecular weight distribution (Blue ®

plus L pre-stained standard cat LC5925, invitrogen). A) Coomassie brilliant blue

R-250 stain of SDSPAGE gel. B) Western blot. Lanes in both gels were loaded as

follows: 1) Collagen VI, 2) Raw skin extract, 3) mixed collagen. ...................... 206

Figure 88: Stratified samples. Left - 1st section cut from surface. Right 6th section

cut from surface containing regions of both enamel layer and the remainder of the

grain layer. The enamel layer is visible as the darker region (white arrow) around

the outside of the section particularly at the top right of the image. The lighter

region (dark arrow) is the remainder of the grain layer that has been uncovered by

the removal of successive sections........................................................................ 211

Figure 89: AFM image of outermost surface of conventionally depilated pickled

lambskin scanned at 20 um resolution. The collagen is visible as single fibrils lying

in a fine mesh. The box marked in white is magnified in Figure 90. ................... 215

Figure 90: Magnification of the AFM image of conventionally depilated skin shown

in Figure 89 showing detail of the fine mesh of individual collagen fibrils. The bar

in the magnified region is 1 um ............................................................................ 215

Figure 91: AFM image of outermost surface of enzymatically depilated pickled

lambskin scanned at 20 um. The collagen is visible as single fibrils lying in a

disorganised pattern. Some fibrils lie enmeshed with others but a significant

number appear loose and independent of the rest of the surface structure. The box

marked in white is magnified in Figure 92. .......................................................... 216

Figure 92: Magnification of the AFM image of enzymatically depilated skin shown

in Figure 91 showing detail of the disorganised loose mesh of collagen fibrils. The

bar in the magnified region is 1 um ...................................................................... 216


xxv

Figure 93: AFM image of the interior of the grain enamel of a conventionally

processed pelt. Note the large grouping of fibrils not seen on the surface on the

grain enamel. .........................................................................................................217

Figure 94: AFM image of the interior of the grain of a conventionally processed

pelt. Again there is a large grouping of fibrils not seen on the surface on the grain

enamel. 218

Figure 95: AFM image of the interior of the grain enamel of an enzymatically depilated

pelt. Note the degree of disorganisation of fibrils in comparison to the

conventionally processed material. .......................................................................218

Figure 96: AFM image of the interior of the grain of an enzymatically depilated

pelt. Note the degree of disorganisation of fibrils in comparison to the

conventionally processed material. .......................................................................219

Figure 97: 2D electrophoretic (8 x 10cm) pattern of the sample sectioned from the

enamel layer of the conventionally depilated skin. Note the spot labelled 1. Spot

one was not identified in material sectioned from the remainder of the grain layer,

indicating it is a protein that is relatively rich in the enamel layer. Spots two and

three represent other proteins that are present in the enamel layer as well as in the

remainder of the grain layers. ...............................................................................220


region of the grain underneath the enamel layer of the conventionally depilated

skin. Note the absence of a spot in the location of spot 1 from Figure 97............221


enamel layer of the conventionally depilated skin. Note the spot labelled 1, which

does not appear in other samples is split, possibly due to cleavage of the peptide or

the partial removal of glycosaminoglycans. The entire spot was sampled for

MALDI TOF analysis. ..........................................................................................221


enamel layer of the enzymatically depilated skin. Note the absence of a spot in the

location of spot 1 from Figure 99. Also note the large number of proteins present in

the enzyme depilated skin extract in comparison to the conventionally depilated

skin. .......................................................................................................................222

Figure 101: Initial experiment for enzyme activity vs. Sodium chloride concentration

response. The error for this test was previously determined to be 0.0018 AU/s or

R.L. Edmonds

xxvi

about 20% of the measured rate (section 3.3.1.4). Note the general reduction in

activity with respect to the addition of sodium chloride. ...................................... 233

Figure 102: Effect of sodium chloride on grain surface examined at 5× magnification

(arrows indicate regions described in Table 38). .................................................. 236

Figure 103: Impact of various salts on enzyme activity against general skin substance

............................................................................................................................... 241

Figure 104: Impact of various salts on grain surfaces .............................................. 244

Figure 105: SACPIC stains of single section depilation micro assay: Note evidence

of the processes of depilation in sections treated with Purafect or Dispase. All bars

are 50 um252

Figure 106: Collagen VI immunohistology of single section depilation micro assay:

Note some collagen VI in each section (black arrows) with evidence of the removal

of stained material within the grain enamel of sections treated with either Dispase

or Purafect (white arrows). All bars are 25 um..................................................... 253


xxvii

Index of tables

Table 1: Glossary ..........................................................................................................xxx

Table 2: conventional sulfide fellmongering constituents ................................................5

Table 3: Composition of hide and skin ...........................................................................32

Table 4: Endogenous enzymes capable of digesting extracellular matrix proteins ........73

Table 5: Enzymes selected for screening ........................................................................82

Table 6: Enzyme application formulation.......................................................................83

Table 7: Enzyme depilation results .................................................................................84

Table 8: Grain surface appearance of tanned skins following enzymatic treatments. ....90

Table 9: Dilution factors used in SDS-PAGE based enzyme characterisation...............93

Table 10: Depilation results for enzymes separated on low pH native PAGE gel .........95

Table 11: Depilation results of "Ornstein Davis" pH 4.2 separation of enzymes...........96

Table 12: SPA error estimation.....................................................................................104

Table 13: Buffers for pH influence on skin powder azure assay ..................................105

Table 14: Grain quality after enzyme wool depilation (Experimental replicate 1).......121

Table 15: Grain quality after enzymatic depilation (Experimental replicate 2)............122

Table 16: Grain quality after enzyme wool loosening (Experimental replicate 3) .......122

Table 17: Rate of enzymatic depilation progression.....................................................129

Table 18: Grain surface assessment descriptive scale...................................................154

Table 19: Results of SACPIC staining during the enzymatic depilation process .........161

Table 20: Alcian blue - Periodic acid Schiff staining results where regions stained blue

contain carboxyl or sulfate groups, regions stained magenta contained material

with glycol units such as sugars and regions stained purple contain both. ...........165

Table 21: Antibodies available for examination of the depilation process ...................169

Table 22: Enzymatically depilated skins; SACPIC stained structural controls. Collagen

stained green, cell nuclei stained black, and the inner root sheath is stained red. 174

Table 23: Immunohistology of fibronectin in enzymatically depilated pelts. ..............190

Table 24: Immunohistology of elastin in enzymatically depilated pelts. .....................192

Table 25: Immunohistology of collagen VI in enzymatically depilated pelts. .............195

Table 26: Immunohistology of collagen VII in enzymatically depilated pelts.............197

Table 27: Immunohistology of laminin in enzymatically depilated pelts.....................200

Table 28: Immunohistology of laminin in enzymatically depilated pelts.....................201

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Table 29: Proteins identified from spots in 2-D gel electrophoresis......................... 223

Table 30: Observed and calculated masses of the tryptic peptides from spot 1 derived

from Figure 97 as matched with UniProtKB/TrEMBL entry name

Q1JQB0_BOVIN.................................................................................................. 223



Q9MZW2_SHEEP................................................................................................ 224


from Figure 97 as matched with UniProtKB/TrEMBL entry name CO3A1_BOVIN

225



Q1JQB0_BOVIN.................................................................................................. 225



Q9MZW2_SHEEP................................................................................................ 226



CO6A2_HUMAN................................................................................................. 226

Table 36: Final NaCl concentrations used with the different enzyme formulations .... 234

Table 37: Enzyme effect on ease of depilation and wool removal ............................... 235

Table 38: Effect of sodium chloride on grain surface................................................... 237

Table 39: Grain flattening effect ................................................................................... 238

Table 40: Effect of enzyme depilation in the presence of salt on grain microstructures

............................................................................................................................... 239

Table 41: Salt concentrations imparting 20% reduction in activity against skin powder

azure (SPA) ........................................................................................................... 242

Table 42: Impact of various salts on depilation ............................................................ 243


xxix

Preface: Thesis overview

In order to contribute to the knowledge of enzymatic depilation of ovine pelts the

following work is reported:

• Chapter one entails: A definition of the problem at hand, the requirements

for work done in this thesis, and describes the approach taken for this work.

• Chapter two contains a detailed description of conventional lime sulfide

based depilation and its associated problems; a description of the possible

alternatives to lime sulfide based depilation and how they relate to enzyme

depilation; a detailed description of the structure of the skin and the bio-

chemicals in the skin; and a description of the current understanding of the

mechanism of enzymatic depilation.

• Chapter three includes an evaluation of the barriers to a successful enzymatic

depilation process and a determination is made of what work was required to

understand the enzyme depilation mechanism and so enable the prediction of

a successful enzymatic depilation process.

• Chapter four builds on conclusions from chapter three and assesses the issue

of variability in depilation across the skin by investigations of heterogeneity

in the skin, thickness and diffusion through the skin, and skin composition.

• Chapter five assesses the protein and protease requirements for the

production of high quality leather from enzyme depilated material including

an assessment of the literature and analysis of protein changes in enzyme

depilated skin compared with conventionally depilated skin. Parts of this

chapter have been published in (Allsop et al., 2007) and (Edmonds et al.,

2008). Immunohistology incorporating cross sections of lambskin exposing

all the different layers described in chapter 2 was used extensively in this

section

• Chapter six investigates the possibility of controlling the adverse effects

noted during enzymatic depilation through the use of salts, and the use of

targeted enzyme reagents.

• Chapter seven concludes this thesis, discusses the findings and their

implications in enzymatic depilation and suggests possible new directions of

research.

R.L. Edmonds

xxx

Glossary

The following glossary was prepared from a selection of reference sources ((Roether,

1976),(van der Loo et al., 1999), (Harris, 1974))

Table 1: Glossary

word description

Bate

Reagent applied during bating, usually an enzyme product such as pancreatic trypsin. (n)

The process by which interfibrilar proteins are removed and the collagen structure opened up usually carried out after liming at pH 9

and 35°C. (v)

Beamhouse processing

The process of loosening wool or hair from skins and removing non-collagenous proteins from the hide or skin and preserving the skin through a pickling process in such a way that it is ready for tannage.

Eg see “conventional processing”

Conventional processing

In the context of this thesis conventional processing is the lime-sulfide based process whereby raw skins are painted with a solution

containing hydrated lime, and sodium sulfide, which loosens the wool. The pulled slats are then limed, delimed, bated, and pickled.

Crust A skin that has been tanned retanned, dyed, and dried but not yet

finished.

Delime The process by which the pH of a slat is brought down from around 12 or 13 down to 8 or 9. This is usually carried out in a processing vessel using a deliming agent such as carbon dioxide or ammonium sulfate.

Depilation see "Dewool".

Dewool The process by which skins are treated so as to loosen the wool to an

extent that it can be removed easily from the skin.

Drum A rotating vessel in which skins are processed. (n)

Apply mechanical action in a drum through rotation of the vessel. (v)

Enzyme A protein which acts as a catalyst in chemical reactions especially

lysis reactions.

Fell Describes the skin of an animal as it is removed after slaughter.

Fellmongery The place at which raw skins are processed to remove wool or hair and other unwanted proteins from a skin as it is converted into a

pickle.

Finish

A coating applied to the surface of a finished leather. (n)

The process of applying a coating to crust leather in order to generate finished leather. (v)

Finished leather

Leather that is ready for use in a leather object such as a garment or as upholstery for example.

Flesh

The side of the skin that was inside the animal. (n)

The process of removing excess fat and muscle from the flesh side of the skin prior to processing. (v)

Float

The liquid in the process vessel in which skins and reagents are drummed. (n)

To add liquid to a drum to achieve a desired liquid level. (v)


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Grain The outermost layer of skin remaining after successful processing to

crust leather.

Green skin A skin immediately after its removed from animal at slaughter (also

known as raw skin).

Hydro extract

In the context of this work this process involves the use of the LASRA hydro extractor which spins the skins at a constant rate for a constant

amount of time and produces a skin with a consistent quantity of associated water.

Liming The process in which the residual wool and soluble proteins and glycosaminoglycans are removed from the skin after the bulk of

loosened wool has been removed.

Opening up The process by which collagen fibres are separated during

processing.

Official sampling

position (OSP)

A position on a skin near the back bone and about one third of the way from the butt to the neck. (IUP2, 2000)

Paint

A solution containing depilatory reagents usually applied in a thickened form to the flesh side of a skin. (n)

The process of applying a depilatory paint usually to the flesh side of a skin. (v)

Pickle

An intermediate stage in the process of converting raw skins into leather when the skin is preserved in a highly acid state in the

presence of salt. (n)

The process of salting and acidifying a skin from which the unwanted proteins have be removed. (v)

Potting The period during which the skins are held after they have been

painted with depilatory during which the chemicals penetrate the skin.

Pre-fleshing See flesh (v).

Protease An enzyme that catalyses the hydrolysis of proteins.

Pulling The process by which the wool is removed from the skin after it has

been loosened by a depilatory.

Shrinkage The thermal denaturation of collagen resulting in a change in

morphology of the collagen fibres with an associated loss in length.

Slat The skin after the loosened wool has been removed but before any

processing has been carried out.

Slip Loosened hair or wool.

Wet blue A skin that has been tanned with chromium but is still wet and has not

yet been retanned or dyed.

Wetting back The process of taking a salted skin or dry pickled pelt and drumming with liquid in order to rehydrate the skin so it can be further processed.

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